Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H(2)O(2) were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H(2)O(2) in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H(2)O(2) via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species

Acute cessation of flow (ischemia) leads to depolarization of the endothelial cell (EC) membrane mediated by K_ATP channels and followed by production of reactive oxygen species (ROS) from NADPH oxidase. We postulated that ROS are a signal for initiating EC proliferation associated with the loss of shear stress. Flow cytometry was used to identify proliferating CD31-positive pulmonary microvascular endothelial cells (mPMVECs) from wild-type, Kir6.2^–/–, and gp91^phox–/– mice. mPMVECs were labeled with PKH26 and cultured in artificial capillaries for 72 h at 5 dyn/cm² (flow adaptation), followed by 24 h of stop flow or continued flow. ROS production during the first hour of ischemia was markedly diminished compared with wild-type mice in both types of gene-targeted mPMVECs. Cell proliferation was defined as the proliferation index (PI). After 72 h of flow, >98% of PKH26-labeled wild-type mPMVECs were at a single peak (PI 1.0) and the proportion of cells in the S+G₂/M phases were at 5.8% on the basis of cell cycle analysis. With ischemia (24 h), PI increased to 2.5 and the ratio of cells in S+G₂/M phases were at 35%. Catalase, diphenyleneiodonium, and cromakalim markedly inhibited ROS production and cell proliferation in flow-adapted wild-type mPMVECs. Significant effects of ischemia were not observed in Kir6.2^–/– and gp91^phox–/– cells. ANG II activation of NADPH oxidase was unaffected by K_ATP gene deletion. Thus loss of shear stress in flow-adapted mPMVECs results in cell division associated with ROS generated by NADPH oxidase. This effect requires a functioning cell membrane K_ATP channel. cell signaling; ischemia; mechanotransduction; K_ATP channels; NADPH oxidase     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

NADPH oxidase subunit p22phox-mediated reactive oxygen species contribute to angiogenesis and tumor growth through AKT and ERK1/2 signaling pathways in prostate cancer

Excessive generation of reactive oxygen species (ROS) in cancer cells is associated with cancer development, but the underlying mechanisms and therapeutic significance remain elusive. In this study, we reported that levels of ROS and p22^phox expression are greatly increased in human prostate cancer tissues, and knockdown of p22^phox by specific small interfering RNA (siRNA) decreased ROS levels in prostate cancer cells. We also showed that stable downregulation of p22^phox in prostate cancer cells inhibited cell proliferation and colony formation, which was mediated by AKT and extracellular signal-regulated kinase (ERK)1/2 signaling pathways and their downstream molecules hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). The NADPH oxidase subunit NOX1 was also elevated in prostate cancer cells, and was involved in activation of AKT/ERK/HIF-1/VEGF pathway and regulation of cell proliferation. Knockdown of p22^phox resulted in inhibition of tumor angiogenesis and tumor growth in nude mice. These findings reveal a new function of p22^phox in tumor angiogenesis and tumor growth, and suggest that p22^phox is a potential novel target for prostate cancer treatment.     

LMP1 Increases Expression of NADPH Oxidase (NOX) and Its Regulatory Subunit p22 in NP69 Nasopharyngeal Cells and Makes Them Sensitive to a Treatment by a NOX Inhibitor

Oxidative stress is thought to contribute to cancer development. Epstein–Barr virus (EBV) and its encoded oncoprotein, latent membrane protein 1 (LMP1), are closely associated with the transformation of nasopharyngeal carcinoma (NPC) and Burkitt’s lymphoma (BL). In this study, we used LMP1-transformed NP cells and EBV-related malignant cell lines to assess the effects of LMP1 on reactive oxygen species (ROS) accumulation and glycolytic activity. Using NPC tissue samples and a tissue array to address clinical implications, we report that LMP1 activates NAD(P)H oxidases to generate excessive amount of ROS in EBV-related malignant diseases. By evaluating NAD(P)H oxidase (NOX) subunit expression, we found that the expression of the NAD(P)H oxidase regulatory subunit p22^phox was significantly upregulated upon LMP1-induced transformation. Furthermore, this upregulation was mediated by the c-Jun N-terminal kinase (JNK) pathway. In addition, LMP1 markedly stimulated anaerobic glycolytic activity through the PI3K/Akt pathway. Additionally, in both NPC cells and tissue samples, p22^phox expression correlated with LMP1 expression. The NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) also exerted a marked cytotoxic effect in LMP1-transformed and malignant cells, providing a novel strategy for anticancer therapy.     

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification,apoptosis, and localized lipid peroxidation

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O₂^?-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91^phox and p22^phox) forming the catalytic core, three cytosolic proteins (p67^phox, p47^phox and p40^phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called ‘Trimera’, composed of the essential domains of the cytosolic proteins p47^phox (aa 1–286), p67^phox (aa 1–212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.     

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91^phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47^phox was translocated to the cell membrane and localized with p22^phox and gp91^phox to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.     

NADPH oxidase in human lung fibroblasts

Reactive oxygen species produced by NADPH oxidase appear to play a role in the response of human lung fibroblast cells to rhinovirus infection. The purpose of the following studies was to characterize the NADPH oxidase components in these cells, to examine the effect of rhinovirus challenge on the expression of these proteins, and to confirm previous studies suggesting a role for p47-phox in the oxidant response to rhinovirus challenge. The results revealed that the NADPH oxidase components p47-phox, p67-phox, p22-phox, and NOX4 were expressed in lung fibroblast cells. In contrast, gp91-phox was not expressed in this cell line. Expression of p67-phox was upregulated by rhinovirus challenge. The functional role of NADPH oxidase in the rhinovirus-induced oxidant stress and elaboration of IL-8 was confirmed by detection of significant reductions in oxidant stress and IL-8 elaboration following transfection of the cells with antisense nucleotides to p47-phox. The lack of gp91-phox in cultured lung fibroblast cells, the induction of p67-phox by rhinovirus, and the confirmation of participation of p47-phox in rhinovirus-induced oxidant stress are significant findings of this study and form a basis for future investigations into understanding the mechanisms of the NADPH oxidase response to rhinovirus infection.     

Expression of NADPH oxidase homologues and accessory genes in human cancer cell lines,tumours and adjacent normal tissues

The family of NADPH oxidase (NOX) genes produces reactive oxygen species (ROS) pivotal for both cell signalling and host defense. To investigate whether NOX and NOX accessory gene expression might be a factor common to specific human tumour types, this study measured the expression levels of NOX genes 1–5, dual oxidase 1 and 2, as well as those of NOX accessory genes NoxO1, NoxA1, p47^phox, p67^phox and p22^phox in human cancer cell lines and in tumour and adjacent normal tissue pairs by quantitative, real-time RT-PCR. The results demonstrate tumour-specific patterns of NOX gene expression that will inform further studies of the role of NOX activity in tumour cell invasion, growth factor response and proliferative potential.     

NADPH Oxidase-generated Hydrogen Peroxide Induces DNA Damage in Mutant FLT3-expressing Leukemia Cells

Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22^phox, a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22^phox and p22^phox-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22^phox, and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H₂O₂)-specific dye, peroxy orange 1 (PO1), and nuclear H₂O₂, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H₂O₂ following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22^phox localize to the nuclear membrane in MV4–11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22^phox mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability.     

NAD(P)H oxidase and eNOS play differential roles in cytomegalovirus infection-induced microvascular dysfunction

Primary cytomegalovirus (CMV) infection promotes oxidative stress and reduces nitric oxide (NO) bioavailability in endothelial cells. These events are among the earliest vascular responses to cardiovascular risk factors. We assessed the roles of NAD(P)H oxidase and NO bioavailability in microvascular responses to persistent CMV infection alone or with hypercholesterolemia. Wild-type (WT) or gp91^phox (NAD(P)H oxidase subunit) knockout mice received mock inoculum or 3 × 10⁴ PFU murine CMV (mCMV) ip 5 weeks before placement on a normal or high-cholesterol diet (HC) for 4 weeks before assessment of arteriolar function and venular blood cell recruitment using intravital microscopy. Some WT groups received sepiapterin (a precursor of the nitric oxide synthase cofactor tetrahydrobiopterin) or apocynin (NAD(P)H oxidase inhibitor/antioxidant). Endothelium-dependent vasodilation was impaired in mCMV vs mock WT, regardless of diet. This was not affected by sepiapterin, and pharmacological inhibition of nitric oxide synthase reduced dilation similarly in mock and mCMV mice. Apocynin or deficiency of total, but not blood cell or vascular wall only (tested using bone marrow chimeras), gp91^phox protected against arteriolar dysfunction. Blood cell recruitment was induced by mCMV–HC. Sepiapterin, but not NAD(P)H oxidase deficiency/apocynin, reduced leukocyte accumulation, whereas platelet adhesion was reduced by sepiapterin, apocynin, or total, platelet-specific, or vascular wall gp91^phox deficiency. These data implicate activation of both hematopoietic and vessel wall NAD(P)H oxidase in mCMV-induced arteriolar dysfunction and platelet and vascular NAD(P)H oxidase in the thrombogenic phenotype induced by mCMV–HC. In contrast, findings with sepiapterin suggest that eNOS dysfunction, perhaps uncoupling, mediates venular, but not arteriolar, responses to mCMV–HC, thus indicating that NAD(P)H oxidase and eNOS differentially regulate microvascular responses to mCMV.     

NADPH-oxidase activation and cognition in Alzheimer disease progression

Superoxide production via NADPH-oxidase (NOX) has been shown to play a role in a variety of neurological disorders, including Alzheimer disease (AD). To improve our understanding of the NOX system and cognitive impairment, we studied the various protein components of the phagocytic isoform (gp91^phox, or NOX2) in the frontal and temporal cortex of age- and postmortem-matched samples. Individuals underwent antemortem cognitive testing and postmortem histopathologic assessment to determine disease progression and assignment to one of the following groups: no cognitive impairment (NCI), preclinical AD, mild cognitive impairment (MCI), early AD, and mild-to-moderate AD. Biochemical methods were used to determine overall NOX activity as well as levels of the various subunits (gp91^phox, p67^phox, p47^phox, p40^phox, and p22^phox). Overall enzyme activity was significantly elevated in the MCI cohort in both cortical regions compared to the NCI cohort. This activity level remained elevated in the AD groups. Only the NOX cytosolic subunit proteins (p67^phox, p47^phox, and p40^phox ) were significantly elevated with disease progression; the membrane-bound subunits (gp91^phox and p22^phox) remained stable. In addition, there was a robust correlation between NOX activity and the individual's cognitive status such that as the enzyme activity increased, cognitive performance decreased. Collectively, these data show that upregulated NADPH-oxidase in frontal and temporal cortex suggests that increases in NOX-associated redox pathways might participate in early pathogenesis and contribute to AD progression.     

Role of VPO1, a newly identified heme-containing peroxidase, in ox-LDL induced endothelial cell apoptosis

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91^phox subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91^phox subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91^phox subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis.     

Methamphetamine alters occludin expression via NADPH oxidase-induced oxidative insult and intact caveolae

Methamphetamine (METH) is a drug of abuse with neurotoxic and vascular effects that may be mediated by reactive oxygen species (ROS). However, potential sources of METH-induced generation of ROS are not fully understood. This study is focused on the role of NAD(P)H oxidase (NOX) in METH-induced dysfunction of brain endothelial cells. Treatment with METH induced a time-dependent increase in phosphorylation of NOX subunit p47, followed by its binding with gp91 and p22, and the formation of an active NOX complex. An increase in NOX activity was associated with elevated production of ROS, alterations of occludin levels and increased transendothelial migration of monocytes. Inhibition of NOX by NSC 23766 attenuated METH-induced ROS generation, changes in occludin protein levels and monocyte migration. Because an active NOX complex is localized to caveolae, we next evaluated the role of caveolae in METH-mediated toxicity to brain endothelial cells. Treatment with METH induced phosphorylation of ERK1/2 and caveolin-1 protein. Inhibition of ERK1/2 activity or caveolin-1 silencing protected against METH-induced alterations of occludin levels. These findings indicate an important role of NOX and functional caveolae in METH-induced oxidative stress in brain endothelial cells that contribute to the subsequent alterations of occludin levels and transendothelial migration of inflammatory cells.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

A novel superoxide-producing NAD(P)H oxidase in kidney

During phagocytosis, gp91(phox), the catalytic subunit of the phagocyte NADPH oxidase, becomes activated to produce superoxide, a precursor of microbicidal oxidants. Currently increasing evidence suggests that nonphagocytic cells contain similar superoxide-producing oxidases, which are proposed to play crucial roles in various events such as cell proliferation and oxygen sensing for erythropoiesis. Here we describe the cloning of human cDNA that encodes a novel NAD(P)H oxidase, designated NOX4. The NOX4 protein of 578 amino acids exhibits 39% identity to gp91(phox) with special conservation in membrane-spanning regions and binding sites for heme, FAD, and NAD(P)H, indicative of its function as a superoxide-producing NAD(P)H oxidase. The membrane fraction of kidney-derived human embryonic kidney (HEK) 293 cells, expressing NOX4, exhibits NADH- and NADPH-dependent superoxide-producing activities, both of which are inhibited by diphenylene iodonium, an agent known to block oxygen sensing, and decreased in cells expressing antisense NOX4 mRNA. The human NOX4 gene, comprising 18 exons, is located on chromosome 11q14.2-q21, and its expression is almost exclusively restricted to adult and fetal kidneys. In human renal cortex, high amounts of the NOX4 protein are present in distal tubular cells, which reside near erythropoietin-producing cells. In addition, overexpression of NOX4 in cultured cells leads to increased superoxide production and decreased rate of growth. The present findings thus suggest that the novel NAD(P)H oxidase NOX4 may serve as an oxygen sensor and/or a regulator of cell growth in kidney.